Vascular endothelial growth factor (VEGF) is a heparin-binding growth factor specific to vascular endothelial cells, which is capable of inducing angiogenesis in vivo. Human VEGF protein was successfully purified and identified by American scientists in 1989, who also cloned and determined its gene sequence.
VEGF is capable of promoting angiogenesis. All members of the VEGF family are capable of activate cell reaction through corresponding receptors (VEGFRs) on the surface of binding cells, and are dimerized and activated through phosphorylation. VEGFRs comprise 7 immunoglobulin-like extracellular domains, one membrane-spanning domain and one intracellular domain that comprises tyrosine kinase domain. VEGF A is capable of binding with VEGF receptor 1 (receptor Flt-1) and VEGF receptor 2 (KDR/Flk-1). VEGF receptor 2 mediates almost all known biological functions of VEGF. VEGF and bioactivity and receptors thereof have been explained and studied in detail by Matsumoto et al. and Marti et al. (see Angiogenesis in ischemic disease. Thromb Haemost. 1999 Suppl 1: 44-52; VEGF receptor signal transduction Sci STKE. 2001: RE21).
VEGF is a highly conserved homodimeric glycoprotein, in which two single strands each having molecular weight of 24 kDa form the dimer through disulfide bond. Because of different ways of splicing by mRNA, at least 5 protein patterns, including VEGF 121, VEGF 145, VEGF 165, VEGF 185 and VEGF206, are developed, wherein VEGF121, VEGF 145 and VEGF 165 are secretory soluble proteins capable of directly acting on vascular endothelial cells, promoting proliferation and migration of vascular endothelial cells, and enhancing vascular permeability.
VEGF-related diseases typically have the following characteristics: over-proliferation of vascular endothelial cells, increase of vascular permeability, tissue edema and inflammation, such as cerebral edema caused by injuries, stroke or tumor; edema caused by inflammatory diseases, such as psoriasis or arthritis, including rheumatoid arthritis; asthma; anasarca associated with burns; ascites and pleural effusion caused by tumor, inflammation or trauma; chronic tracheitis; capillary leak syndrome; sapremia; kidney diseases associated with protein leakage; eye diseases, such as age-related macular degeneration and diabetic retinopathy; tumors, including breast cancer, lung cancer, colorectal cancer, brain glioma, kidney cancer, etc.
The binding between the antibody and its target spot is specific, which can mediate immunological effect mechanism and has relatively long half-life in serum. These characteristics result in strong treatment applications of the antibody.
Currently, FDA and Europe have approved the application of recombinant humanized mouse anti-VEGF monoclonal antibody, AVASTIN, in treating colorectal cancer, non-small cell lung cancer, breast cancer, brain glioma, kidney cancer and age-related macular degeneration (AMD), which reached sales of 4.8 billion US dollars in 2008. However, the AVASTIN antibody does not have a high affinity to VEGF. Because of the exclusive production, moreover, patients need to pay high prices. Currently, a patient needs to pay between about 50,000 and 100,000 US dollars per year for the drug. Therefore, there is an urgent need to develop new anti-VEGF monoclonal antibodies, thereby reducing loads on patients and lowering treatment costs.
Definitions
Prior to further description of the present invention, it is necessary to understand that the present invention is not limited by the described specific embodiments. In other words, variations may be made in specific formats. It should be further noted that since the scope of the present invention is subject to the appended claims, terms herein are only intended to describe the specific embodiments, instead of limiting the present invention.
Terms “antibody” and “immunoglobulin” may be used interchangeably herein. These terms are well known to those skilled in the art and specifically refer to proteins consisted of one or more polypeptides capable of specifically binding with antigens. One form of the antibody constitutes a basic structural unit of the antibody, which is tetramer. It consists of two pairs of completely identical antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, variable domains of the light chain and the heavy chain are joined together to be responsible for binding with antigens, while the constant domains are responsible for effector functions of the antibody.
Currently known immunoglobulin polypeptides comprise κ and λ light chains, and α, γ (IgG1, IgG2, IgG3, IgG4), δ, ε and μ heavy chains or other equivalents thereof. The immunoglobulin “light chain” (about 25 kDa or about 214 amino acids) in its whole length comprises a variable domain consisted of about 110 amino acids at the NH2-terminal, and a κ or λ constant domain at the COOH-terminal. Similarly, the immunoglobulin “heavy chain” (about 50 kDa or about 446 amino acids) in its whole length comprises a variable domain (about 116 amino acids) and one of heavy chain constant domains, such as γ (about 330 amino acids).
Terms “antibody” and “immunoglobulin” comprise any isoform antibodies or immunoglobulins, or antibody segments that are still specifically bound with antigens, including but not limited to Fab, Fv, scFv and Fd segments, chimeric antibody, humanized antibody, single-strand antibody, as well as fusion proteins having antigen binding portions of antibodies and non-antibody proteins. Antibodies may be labeled and detected, for example, by radioactive isotopes, enzymes capable of producing assayable substances, fluorescent proteins and biotins. Furthermore, antibodies can bind with solid carriers, including but not limited to polystyrene plates or beads. Said term further comprises Fab′, Fv, F(ab′)2 and/or other antibody segments and monoclonal antibodies capable of specifically binding with antigens.
Antibodies may also exist in a variety of forms, for example, comprising Fv, Fab and (Fab)2, as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol, 1987; 17, 105), and in the form of single strand (e.g., Huston et al., Proc. NatL Acad. Sci. U.S.A., 1988; 85, 5879 and Bird et al., Science, 1988; 242, 423, which are cited herein as reference). Variable domains of heavy chain or light chain of immunoglobulin consist of three hypervariable domains (also referred to as “complementarity determining region” or CDR). These hypervariable domains are spaced apart by framework regions (FR). The scopes of FR and CDR have been precisely defined (see “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, 1991). Amino acid sequences of all antibodies discussed herein are sorted by referring to the Kabat system. Different light chain and heavy chain FR sequences of the same species are relatively conserved. Antibody FRs are used to position and calibrate CDRs. CDRs are mainly responsible for binding with antigen epitopes.
A chimeric antibody is an antibody with constructed heavy chain and light chain genes, in particular an antibody with variable domain and constant domain genes that are genetically engineered and belong to different species. For example, variable domain segments of mouse monoclonal antibody genes are joined to constant domain segments of human antibody, such as γ1 and γ3. For example, chimeric antibodies used in medical treatment are a type of chimeric proteins, which are rabbit antibody variable domain segments or antigen binding domain segments combined with human antibody constant domains or effect domains (e.g. the anti-Tac chimeric antibody prepared with the cell deposited under the accession number of A.T.C.C. No. CRL 9688). Of course, chimeric antibodies can also use genes from other mammal species.
Terms “humanized antibody” and “humanized immunoglobulin” have the same meaning. Compared with the non-humanized form of an antibody, its humanized antibody typically reduces the immunoreaction in the human host.
It should be understood that the humanized antibody designed and produced according to the present invention may replace some conservative amino acids, which have substantially no impact on antigen binding or other functions of the antibody. In other words, amino acids can be mutually substituted in the combinations of gly and ala; val, ile and leu; asp and glu; asn and gln; ser and thr; lys and arg; phe and tyr. Amino acids not in the same group are “substantially different” amino acids.
In some embodiments, the affinity between an antibody and its target spot is represented by Kd (dissociation constant), which is lower than 10−6 M, 10−7 M, 10−8 M, 10−9 M, 10−10 M, 10−11 M or about 10−12 M or lower.
“Variable domain” of an antibody's heavy chain or light chain is the mature region at the N terminal of said chain. All domains, CDRs and residue numbers are defined through sequence alignment and based on existing structural knowledge. Determination and numbering of FR and CDR residues are based on what Chothia and others have described (Chothia, Structural determinants in the sequences of immunoglobulin variable domain. J Mol Biol. 1998; 278, 457).
VH is the variable domain of the antibody's heavy chain. VL is the variable domain of the antibody's light chain, which may comprise κ and λ isotypes. K-1 antibody has the κ-1 isotype, while K-2 antibody has the κ-2 isotype, and Vλ is the variable λ light chain.
Terms “polypeptide” and “protein” may be used interchangeably herein. Both of them refer to polymerized amino acids of any length, which may comprise encoding and non-encoding amino acids, chemically or biochemically modified or derived amino acids and polypeptides having modified peptide skeletons. Said terms comprise fusion proteins, including but not limited to fusion proteins having heterogeneous amino acid sequences; fusion proteins having heterogeneous and homogeneous leader sequences, with or without N-terminal methionine residues; proteins with immunological tags; fusion proteins with detectable fusion partners, for example, fusion proteins that can function as fusion partners, including fluorescent protein, β-galactosidase, fluorescein, etc. Polypeptides can be of any length, and the term “peptide” refers to polypeptides of the length of 8-50 residues (e.g. 8-20 residues).
Terms, “subject”, “host”, “patient” and “individual”, may be used interchangeably herein, and specifically refer to any mammals, in particularly humans, that are diagnosed or treated. Other subjects may comprise monkey, cow, dog, cat, Guinea pig, rabbit, rat, mouse, horse, etc.
“Corresponding amino acids” refer to amino acid residues at the same positions (i.e. they correspond to each other) when two or more amino acid sequences are compared. Comparison and numbering methods of antibody sequences have been described in detail by Chothia (see above), Kabat (see above), and others. It is known to those skilled in the art (see, for example, Kabat 1991 Sequences of Proteins of Immunological Interest, DHHS, Washington, D.C.) that sometimes one, two or three gaps may be made, and/or 1, 2, 3 or 4 residues or at most about 15 residues (in particular in L3 and H3 CDRs) may be inserted in one or two amino acids of an antibody, thereby completing a comparison.
“Substitutable position” refers to a special position of an antibody, which can be substituted by different amino acids without significantly reducing the antibody's binding activity. Methods to determine substitutable positions and how they can be substituted will be described below in more detail. The substitutable position may also be referred to as “variation tolerant position”.
“Parent” antibody refers to the target antibody of amino acid substitution. In some embodiments, a “donor” antibody will “donate” amino acids to a parent antibody to produce a changed antibody. “Associated antibody” refers to an antibody having similar sequence and produced by cells having the common B cell ancestor. This B cell ancestor comprises genome that has rearranged light chain VJC domains and rearranged heavy chain VDJC domains, and moreover, produces antibodies that have not experienced affinity maturation. The “naive” or “primary” B cell that exists in the spleen tissue is the common ancestor of B cells. The bindings of associated antibodies with an identical epitope are typically very similar in sequence, in particular their L3 and H3 CDRs. All H3 and L3 CDRs of associated antibodies have the same length and nearly identical sequence (with 0-4 different amino acid residues). Associated antibodies are correlated through the common antibody ancestor, i.e. the antibody produced by the original B cell ancestor.